| Assay Method Information | |
| | Inhibitory Activity against Enzyme hFXIa (Human Factor XIa) |
| Description: | Experimental method:1) Preparation for compoundsAll test compounds were prepared using Echo, subjected to gradient dilution and transferred to a 384-well reaction plate at 100 nL. The reference compound and the test samples, with an initial concentration of 200 uM, were subjected to 3-fold dilution to get 10 concentrations (the final concentration was 1 uM). To high signal wells were added DMSO, and to low signal wells were added 100 uM reference compound. Reference can be made to the sample distribution for distributions of samples and control.1) Preparation for enzyme human factor XIaEnzyme human factor XIa was diluted to 0.5 ug/mL with assay buffer.3) Preparation for substrate S-2306S-2306 was prepared at a concentration of 1 mM using the assay buffer.4) 10 uL of 0.5 ug/mL enzyme human factor XIa was added to the reaction plate using a Mutidrop automatic dispenser and the final concentration was 5 ng/well.5) 10 uL of 1 mM S-2306 was added to the reaction plate using a Mutidrop automatic dispenser and the final concentration was 0.5 mM.6) Centrifugation was performed at 1000 rpm for 10 s.7) The reaction plate was incubated in SpectraMax 340PC at 37 C. for 10 min and the absorbance was measured at 405 nm.8) Data were analyzed using GraphPad Prism 5.0.% inhibition rate=100%x[1 (sample reading mean of low signal wells)/(mean of high signal wells mean of low signal wells)] |
| Affinity data for this assay | |
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